Chlamydial trachomatis are obligate intracellular bacteria and are the most common cause of sexually transmitted diseases (STD) of humans. A common sequelae of genital infection of women is salpingitis that can lead to pelvic inflammatory disease (PID) and infertility. Treatment of chlamydial STDs is estimated to cost 5 billion dollars annually. Although much progress has been made in understanding the pathogenesis of chlamydial STD there remains key unanswered questions in the pathophysiology and immunology of infection that have hampered advances in the development of much needed preventive therapies. A major obstacle toward this end has been the inability to use molecular genetic approaches to study this important human pathogen. It is likely that significant progress in the control of chlamydial STD will only come when genetic manipulation of the pathogen is possible. Recent advances in genomics have greatly increased the feasibility of developing new strategies for the study of many pathogenic microorganisms. This is indeed true for Chlamydia. For example, the availability of the complete C. trachomatis genomic sequence shows that the organism possess a reasonably complete complement of genes necessary for genetic recombination and repair. This strongly suggests that it should be feasible to develop a genetic transformation system for chlamydiae. The purpose of this project is to begin the process of developing a genetic system for Chlamydia. Once the basic tools of genetics are developed the project will address other challenges aimed at defining methods for optimum DNA introduction and transformation. These will include the targeting of both the infectious extracellular and non-infectious intracellular life forms of the organism using electroporation and particle bombardment technologies. Once conditions for stable genetic transformation have been developed experiments will be done to mutate or knock out targeted genes identified from the genome sequence as potentially important virulence determinants, and introduce new genes into the chlamydial genome that might allow the organism to be grown outside cells. The results of these studies should significantly advance the understanding of chlamydial pathogenic mechanism(s) and disease processes, as well as possibly produce insights for the development of novel live-attenuated organisms that can be tested as vaccine candidates in pre-clinical models of chlamydial infection. To date we have used the recently described plaque cloning procedure to isolate spontaneous rifampicin resistant mutants of C. trachomatis. The development of stable genetic resistance markers will enable us to test the ability of C. trachomatis to incorporate DNA following introduction by electroporation or particle bombardment means. - Plaque assay, cloning, antibiotic resistance, recombination, electroporation, particle bombardment, and DNA mutagenesis